Voltage-dependent anion channel localises to the plasma membrane and peripheral but not perinuclear mitochondria

Abstract

Activity of the antioestrogen-activated maxi-Cl(-) channel has been recorded in different cell types, including fibroblasts, vascular smooth muscle, endothelial and neuroblastoma cells. Its electrophysiological properties resemble those of the voltage-dependent anion channel (VDAC) of the outer mitochondrial membrane, a channel of particular relevance to the physiology and pathophysiology of mitochondria. The hypothesis that VDAC could be the molecular correlate of the plasma membrane maxi-Cl(-) channel has been debated over the last few years, with the lack of clear evidence for the presence of VDAC in the plasma membrane constituting the main argument of the detractors. In the present study, we investigated the cellular localisation of VDAC in NIH3T3 fibroblasts. The presence of a plasma membrane VDAC was demonstrated by immunoblotting of membrane fractions with monoclonal antibodies against the VDAC and by RT-PCR using primers that hybridise to a VDAC sequence coding for a N-terminal leader peptide required for its plasma membrane sorting. In addition, confocal microscopy studies showed the colocalisation of VDAC with caveolin-1. As expected, VDAC also localised to mitochondria. Colocalisation studies with TOM-20, a protein also present in the outer mitochondrial membrane, showed that VDAC proteins localised only to peripheral and not to perinuclear mitochondria.

Full text

Pflugers Arch - Eur J Physiol (2003) 446:309–313
DOI 10.1007/s00424-003-1054-7
EJP (PFLGERS ARCHIV)—SYMPOSIUM
Maria I. Bahamonde · Miguel A. Valverde
Voltage-dependent anion channel localises to the plasma membrane
and peripheral but not perinuclear mitochondria
Published online: 16 April 2003
 Springer-Verlag 2003
Abstract Activity of the antioestrogen-activated maxi-
Clchannel has been recorded in different cell types,
including fibroblasts, vascular smooth muscle, endothelial
and neuroblastoma cells. Its electrophysiological proper-
ties resemble those of the voltage-dependent anion
channel (VDAC) of the outer mitochondrial membrane,
a channel of particular relevance to the physiology and
pathophysiology of mitochondria. The hypothesis that
VDAC could be the molecular correlate of the plasma
membrane maxi-Clchannel has been debated over the
last few years, with the lack of clear evidence for the
presence of VDAC in the plasma membrane constituting
the main argument of the detractors. In the present study,
we investigated the cellular localisation of VDAC in
NIH3T3 fibroblasts. The presence of a plasma membrane
VDAC was demonstrated by immunoblotting of mem-
brane fractions with monoclonal antibodies against the
VDAC and by RT-PCR using primers that hybridise to a
VDAC sequence coding for a N-terminal leader peptide
required for its plasma membrane sorting. In addition,
confocal microscopy studies showed the colocalisation of
VDAC with caveolin-1. As expected, VDAC also local-
ised to mitochondria. Colocalisation studies with TOM-
20, a protein also present in the outer mitochondrial
membrane, showed that VDAC proteins localised only to
peripheral and not to perinuclear mitochondria.
Keywords VDAC · Maxi-Clchannel · Lipid rafts ·
Caveolin · Antioestrogen · Mitochondria · Fibroblasts ·
Neuroblastoma
Introduction
Voltage-dependent anion channels (VDACs), also known
as porins, are integral membrane proteins which form a
pore slightly more permeable to anions than cations, and
are also permeable to small solutes [1]. They have been
identified in the mitochondrial outer membrane where
they provide a major pathway for the transport of
metabolites, e.g. ATP [2] and cholesterol [3], among
others. They are also involved in the mitochondrial events
leading to apoptosis [4, 5]. Besides their mitochondrial
location, several reports claim the presence of VDAC in
the plasma membrane of different cell types [6, 7, 8, 9].
The fact that its electrophysiological properties [1]
resemble those of the plasma membrane maxi-Clchan-
nel [10, 11, 12] has encouraged some investigators to
assume that the two channels were one and the same
protein [6, 7, 8, 9, 10, 11, 12, 13]. This hypothesis was
based on early observations suggesting the presence of
VDAC protein on the plasma membrane [7, 8, 9, 10, 11,
12, 13] but has been questioned by others [14]. The extra-
mitochondrial location of VDAC has recently received
strong support from two independent reports: the identi-
fication of a VDAC isoform (pl-VDAC) that contains a
leader sequence for its trafficking to the plasma mem-
brane [9] and the presence of VDAC in caveolae [8].
In the present study, in addition to demonstrating the
plasma membrane location of VDAC and its association
with caveolae in NIH3T3 fibroblasts, we provide the first
evidence suggesting its preferential localisation to a
peripheral pool of mitochondria.
Materials and methods
Cell culture
NIH3T3 mouse fibroblasts permanently transfected with the MDR1
gene were cultured as previously described [11]. When confluent,
cells were either subcultured into T-75 flasks (Nunc) and used
either for immunoblotting or plated on to treated glass cover-slips
for immunofluorescence experiments.
M. I. Bahamonde · M. A. Valverde ())
Unitat de Senyalitzaci Cel·lular,
Departament de Cincies Experimentals i de la Salut,
Universitat Pompeu Fabra, C/Dr. Aiguader 80,
08003 Barcelona, Spain
e-mail: miguel.valverde@cexs.upf.es
Fax: +34-93-5422802

Immunoblotting
Plasma membrane proteins and cell lysate were prepared using a
previously described protocol [15, 16]. Proteins were resolved by
SDS-PAGE (12%) and blotted onto nitrocellulose. The primary
antibody used was the monoclonal mouse anti-porin 31 HL
(VDAC1) (Ab-2 antibody, Calbiochem; 1:2,000 dilution). Non-
specific binding was avoided by incubating the nitrocellulose
membranes in a blocking solution consisting of TTBS buffer
(100 mM TRIS-HCl pH 7.5, 150 mM NaCl) supplemented with 5%
non-fat milk for 1 h at room temperature or overnight at 4C. The
mouse antibody was detected with alkaline-phosphatase-conjugated
antibody (goat anti-mouse IgG, 1:500; Calbiochem). The mem-
branes were then washed and the bands visualised using nitro blue
tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate tolui-
dine salt solution for detection of alkaline phosphatase.
Confocal microscopy
Double-label analysis was carried out on NIH3T3 cells adhered to
12 mm glass cover-slips coated with poly-d-lysine (10 g/ml for
1 h). Cells were fixed in 2% paraformaldehyde, 0.15 M sucrose and
0.1% glutaraldehyde for 10 min and were permeabilised by
incubation with 0.1% Triton X-100 for 10 min. The Triton
treatment was omitted in the case of non-permeabilised cells. Prior
to antibody incubation, cells were treated with NH4Cl for 30 min to
minimise the number of reactive aldehyde groups and blocked for
30 min (room temperature) with 5% fetal bovine serum and 1%
BSA in washing buffer. Cells were then incubated with anti-porin
31 HL (1:80), rabbit anti-caveolin-1 (N20, Santa Cruz Biotechnol-
ogy; 1:1,000) or rabbit anti-Tom20 (FL-145, Santa Cruz Biotech-
nology; 1:100) for 2 h at room temperature. Unbound antibody was
removed by washing the cells 3 times with 1 ml of blocking
solution for 10 min each time. Following several washes in
blocking solution, cells were incubated with secondary Alexa Fluor
488 goat anti-rabbit (Molecular Probes; 1:500) and goat anti-mouse
IgG coupled to the fluorochrome Cy3 (Amersham; 1:1,000) or
sheep anti-mouse Texas Red (Amersham; 1:100), for 1 h at room
temperature. Prior to incubation, the secondary antibody was
centrifuged at 13,000 gfor 15 min at 4 C in order to pellet any
precipitated constituents. Negative controls were performed in
which the cells were solely incubated with the secondary antibody.
Digital images were taken with a Leica TCS SP confocal
microscope and analysed with Leica confocal software (Heidelberg,
Germany).
RNA extraction and RT-PCR
RNA extraction and RT-PCR were performed as described
previously [16]. In brief, RNA was isolated from C1300 cells
using the Nucleospin RNA II kit (Macherey-Nagel, Germany),
according to the manufacturer’s instructions. Total RNA (1 g) was
reverse-transcribed to cDNA. We used the following primer pair for
PCR amplification of pl-VDAC [9]: forward 50-TGTGTTCATTC-
TTTCTCGTGC-30and reverse 50-CCAGTGTTCGGCGAGAAT-
GAC-30. PCR products were analysed on a 2% agarose gel
containing a final concentration of 0.5 g/ml ethidium bromide.
Results and discussion
The recent identification of an alternative exon in the
murine VDAC-1 gene provided evidence of a plasma-
lemmal form of VDAC (pl-VDAC) with an N-terminal
leader sequence for its targeting to the plasma membrane
[9]. The presence of a pl-VDAC form in NIH3T3
fibroblasts was confirmed by RT-PCR using primers
specific to the pl-VDAC, including the leader sequence
(Fig. 1A). A band of the expected size (350 bp) was
identified and confirmed to be pl-VDAC by sequencing.
The presence of VDAC protein on NIH3T3 cells was
tested by Western blot (Fig. 1B). The monoclonal anti-
VDAC antibody used (anti-31HL, in particular Ab-2),
was raised against an N-terminal synthetic peptide [17].
Figure 1B shows a Western blot obtained from a
membrane fraction or whole lysate of NIH3T3 cells. A
single band of ~32 kDa, which is the expected size for
VDAC, was obtained from both preparations, indicating
the absence of cross-reactivity with other cellular pro-
teins. Immunolocalisation of VDAC to the plasma
membrane was confirmed by confocal microscopy
(Fig. 2). Non-permeabilised NIH3T3 cells (Fig. 2B) show
membrane delimited staining, consistent with antibody
recognition of the external N-terminal globular a-helix of
the VDAC protein [18]. Permeabilisation of NIH3T3 cells
(Fig. 2E) resulted in staining of the cytosol, most likely
reflecting its organelle localisation, and a reinforcement
of the membrane signal.
Another piece of evidence suggesting the presence of
VDAC in the plasma membrane has been its isolation
from liquid-ordered membrane microdomains called lipid
rafts and its functional reconstitution in artificial bilayers
[8]. To establish whether VDAC was also present in such
specialised membrane microdomains in NIH3T3 cells, we
carried out colocalisation experiments using confocal
microscopy. An antibody against caveolin-1, a protein
present in structured lipid rafts with flask-shaped mem-
brane invaginations termed caveolae [19, 20], was used.
Figure 3 shows confocal images of NIH3T3 cells probed
with anti-VDAC antibody in red (Fig. 3A), and anti-
caveolin-1 antibody in green (Fig. 3B). The merged image
Fig. 1A, B Voltage-dependent anion channel (VDAC) detection in
NIH3T3 cells. AAliquots (10 g) of plasma membrane proteins or
total protein lysate from NIH3T3 fibroblasts were used to detect
VDAC by Western blot analysis. BRT-PCR analysis of pl-VDAC,
which recognises a band of 350 bp that includes the leader
sequence. For the negative control the cDNA was omitted from the
reaction. Western blot and RT-PCR data are representative of at
least 4–10 different experiments
310

(Fig. 3C) shows a marked overlapping of caveolin-1 and
VDAC at the plasma membrane level. These results
reinforced previous observations suggesting the presence
of VDAC at the plasma membrane and, similar to other
cell types [8], VDAC in NIH3T3 fibroblasts also segre-
gates to the caveolin-1-containing lipid rafts.
As mentioned above, eukaryotic VDAC was first
discovered in the outer membrane of mitochondria [1]
where it forms part of the mitochondrial permeability
transition pore (reviewed in [21]). Mitochondria have
always been seen as a homogeneous population of
organelles, the main task of which is the production of
the cell’s energy [21]. Over the last few years, this view
has changed tremendously. Mitochondria contribute to the
shaping and regulation of Ca2+ signals [22] and mito-
chondrial dysfunction leads to different forms of cell
death [21]. Moreover, it seems that, at least in certain cell
types, different pools of mitochondria exist [23]. There-
fore, we evaluated whether VDAC was uniformly
expressed in NIH3T3 mitochondria (Fig. 4). As a marker
of mitochondria we used an anti-Tom20 antibody. Tom20
is an outer mitochondrial membrane protein that forms
part of the translocation machinery involved in the
recognition and translocation of proteins targeted to the
mitochondria. Tom20 has also been proposed as the
receptor protein responsible for the insertion of VDAC in
the outer membrane of the mitochondria [24]. Overlap-
ping of images obtained with anti-VDAC and anti-Tom20
antibodies (Fig. 4C) revealed a perinuclear pool of
mitochondria positive for Tom20 (green) and a peripheral
pool of mitochondria where VDAC and Tom20 colo-
calised (yellow), surrounded by the red signal provided by
the membrane localisation of VDAC. Such differential
localisation of mitochondrial VDAC has not been report-
ed before. We established whether this pattern is unique
for the NIH3T3 fibroblasts or reflects a more general
pattern. The same colocalisation studies were repeated in
another cell line, the C1300 neuroblastoma cell line, that
also expresses maxi-Clchannel activity in the plasma
membrane [25]. Figure 4F shows an even more vivid
tricolour pattern: green (perinuclear mitochondria stained
with Tom20 antibody), yellow (colocalisation of Tom20
and VDAC in peripheral mitochondria) and red (mem-
brane localisation of VDAC). The existence of different
Fig. 3A–C Localisation of VDAC and caveolin-1-containing lipid
rafts in NIH3T3 cells. APermeabilised NIH3T3 cells immuno-
stained with anti-porin 31 HL (VDAC) antibody and Cy3-coupled
secondary antibody (red). BImmunostaining with anti-caveolin-1
antibody and Alexa Fluor 488 secondary antibody (green). C
Merged image of Aand Bshowing extensive colocalisation of
caveolin-1 and VDAC (yellow). Images were obtained by confocal
laser scanning microscopy and are representative of at least 10
different immunolocalisations
Fig. 2A–F Confocal microscopy imaging of VDAC. Non-perme-
abilised (A,Band C) and permeabilised (D,Eand F) NIH3T3 cells
immunostained with anti-porin 31 HL (VDAC) antibody and Cy3-
coupled secondary antibody (red). Controls (Aand D) were
obtained by incubation solely with the secondary antibody. C,F
Transmitted light images of the cells. They are representative of at
least three different immunolocalisations
311

functional pools of mitochondria has been identified in
chromaffin [22] and pancreatic acinar cells [23]. Based on
the differential expression of Tom20 and VDAC proteins
we now demonstrate that in NIH3T3 fibroblasts and
C1300 neuroblastoma cells, at least two pools of mito-
chondria are present.
VDAC appears to be involved in both apoptotic release
of cytochrome c [5] and Ca2+ signalling [26]. The
relevance of this observation is still far from clear. While
there is no evidence suggesting the involvement of a
specific pool of mitochondria in the events leading to
apoptotic cell death, there are several studies reporting the
presence of a peripheral“ring” of mitochondria which
would protect the inner environment from massive
increases in Ca2+ [22, 23]. It would be interesting to
evaluate the precise part played by the two pools of
mitochondria in apoptotic cell death and to establish
whether there is any link between the plasma membrane
VDAC and mitochondrial VDAC in the events leading to
cell apoptosis.
Acknowledgements This work was supported by the Human
Frontiers Science Program, Spanish Ministry of Science and
Technology, Fondo de Investigaciones Sanitarias and Distinci de
la Generalitat de Catalunya per a la Promoci de la Recerca
Universitria. We thank E. Vazquez and J.M. Fernndez-Fernndez
for critical discussion and A. Currid for proof reading the
manuscript.
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